Swagata Chakraborty

Simultaneous acquisition of 13Cα–15N and 1H–15N–15N sequential correlations in proteins: application of dual receivers in 3D HNN

We describe here, adaptation of the HNN pulse sequence for multiple nuclei detection using two independent receivers by utilizing the detectable 13Cα transverse magnetization which was otherwise dephased out in the conventional HNN experiment. It enables acquisition of 2D 13Cα–15N sequential correlations along with the standard 3D 15N–15N–1H correlations, which provides directionality to sequential walk in HNN, on one hand, and enhances the speed of backbone assignment, on the other. We foresee that the implementation of dual direct detection opens up new avenues for a wide variety of modifications that would further enhance the value and applications of the experiment, and enable derivation of hitherto impossible information.

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein.

Residue-wise conformational stability of DLC8 dimer from native-state hydrogen exchange.

Dynein light chain (DLC8) is the smallest subunit of the dynein motor complex, which is known to act as a cargo adaptor in intracellular trafficking. The protein exists as a pure dimer at physiological pH and a completely folded monomer below pH 4. Here, we have determined the energy landscape of the dimeric protein using a combination of optical techniques and native‐state hydrogen exchange of amide groups, the former giving the global features and the latter yielding the residue level details. The data indicated the presence of intermediates along the equilibrium unfolding transition. The hydrogen exchange data suggested that the molecule has differential stability in its various segments. We deduce from the free energy data that the antiparallel β‐sheets (β4 and β5) that form the hydrophobic core of the protein and the α2 helix, all of which are highly protected with regard to hydrogen exchange, contribute significantly to the initial step of the protein folding mechanism. Denaturant‐dependent hydrogen exchange indicated further that some amides exchange via local fluctuations, whereas there are others which exchange via global unfolding events. Implications of these to cargo adaptability of the dimer are discussed. Proteins 2009.

Selective lighting up of segments around Gly, Ala and Ser/Thr in proteins

Direct detection of 13C nucleus can be used as a valuable alternative where 1H detection poses a challenge due to relaxation effects, chemical exchange and poor chemical shift dispersion. In this context, we have designed a suite of 2D 13Cα‐detected hNCA experiments that provide sequential correlations of 13Cα with 15N on one hand and efficient spectroscopic labeling of certain groups of residues, namely, Gly, Ala, Ser and Thr, on the other. These residues act as checkpoints in the sequential walk, which in turn offer new possibilities of backbone assignment of small proteins from a set of 2D experiments, thereby providing great economy in terms of spectrometer time. The direct identification of peptide segments around Gly, Ala, Ser and Thr residues along a protein chain will be highly valuable for deriving important information on sites of ligand binding, phosphorylation, inhibitor/substrate binding, understanding protein folding pathways, comprehending local conformational dynamics etc. without having to obtain complete sequence‐specific assignments, which can be time consuming and at times formidable, especially in large proteins. We have illustratively demonstrated the multifaceted applications of these variants of 2D experiments on ubiquitin and M‐crystallin. We foresee that these 2D hNCA experiments will provide economic and efficient strategies for studying the structure and function of proteins. Copyright

Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6M urea. NMR studies identified the β3 and β4 strands, N-, C- terminal regions, loops connecting β1 to α1, α1 to α2 and β3 to β4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the proteins multiple functions.

Residual structure and dynamics in DMSO-d6 denatured Dynein Light Chain protein

Structural and motional features in the denatured state of a protein dictate the early folding events starting from that state and these features vary depending upon the nature of the denaturant used. Here, we have attempted to decipher the early events in the folding of Dynein Light Chain protein (DLC8), starting from DMSO-d6 denatured state. Multinuclear NMR experiments were used to obtain the full spectral assignment. The HSQC spectrum shows the presence of two sets of peaks for the residues Met 1, Ser 2, Arg 4, Ala 11, Met 17, Thr 26, Lys 44, Tyr 50, Asn 51, Trp 54, His 55, Val 58, Gly 59, Ser 64, Tyr 65, His 68, Phe 86, Lys 87 indicating the presence of slow conformational transition in the heterogeneous ensemble. Analysis of residual structural propensities with secondary (13)C chemical shifts, (3)J(H(N)(-)H(α)) coupling constants and (1)H-(1)H NOE revealed the presence of local preferences which encompass both native and non-native like structures. The spectral density calculations, as obtained from measured R(1), R(2) and (1)H-(15)N steady state NOE values provide insights into the backbone dynamics on the milli to picosecond timescale. The segment Ser 14 - His 55 exhibits slow motions on the milli- to microsecond timescale arising from conformational exchange. The presence of native like structural preference, as well as conformational exchange classifies the above segment as the nucleation site of folding. Based on the observations, we propose here, the probable hierarchy of folding of DLC8 on dilution of denaturant: the two helices are formed first followed by the formation of β2 and β5.

Resonance assignments of GTPase effector domain of dynamin in the aprotic solvent deuterated dimethyl sulfoxide

The GTPase effector domain (GED) is a subunit of dynamin, a multi-domain protein involved in endocytosis. GED forms a megadalton-sized self-assembly in vitro. The core of such huge assemblies is inaccessible to detailed Nuclear Magnetic Resonance characterization by conventional methods due to line broadening effects. Till date, there have been no studies to directly identify the residues involved in the core of the assembly. In this background we report here the NMR resonance assignments of deuterated dimethyl sulfoxide (DMSO-d6)-denatured GED from Homo sapiens. This will form the basis for probing the core of GED assembly and characterization of the association pathway driven by DMSO dilution.

Intrinsic vs environment driven equilibrium folding transitions in GTPase effector domain of dynamin: NMR insights.

Relative importance of the intrinsic properties of the polypeptide chain vis-a-vis the environmental influences, in driving the folding of a protein, has been a subject of extensive debate and investigation. Folding/misfolding lead to self association in many systems, which have biological functional significance. We compare here, the NMR derived equilibrium folding transitions driven under different environmental conditions in the GTPase Effector Domain of dynamin, which self-associates into megadalton size species. We conclude that though hierarchy of folding and association of GED is substantially influenced by the solvents, these properties, to a good extent are also driven by intrinsic properties of the polypeptide chain, and the regions that form secondary structures, the types of secondary structures formed in those regions, and finally the regions that participate in the self-association are the same, indicating near neighbor interactions would have a telling effect on the final outcome of the folding process. These observations would open a new reliable frontier for elucidating the multiple folding trajectories and consequent self-association, by simulations in vacuum, for this protein.

NMR Derived Model of GTPase Effector Domain (GED) Self Association: Relevance to Dynamin Assembly

Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its ‘coiled coil’ GTPase Effector Domain (GED), which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the self-association of GED by first dissociating the assembly using Dimethyl Sulfoxide (DMSO) and then systematically monitoring the refolding into helix and concomitant re-association using NMR spectroscopy, as DMSO concentration is progressively reduced. The short segment, Arg109 - Met116, acts as the nucleation site for helix formation and self-association. Hydrophobic and complementary charge interactions on the surfaces drive self-association, as the helices elongate in both the directions resulting in an antiparallel stack. A small N-terminal segment remains floppy in the assembly. Following these and other published results on inter-domain interactions, we have proposed a plausible mode of dynamin self assembly.